1. Field of the Invention
The present invention generally relates to ricin vaccines and methods of making and using thereof.
2. Description of the Related Art
Ricin is a very toxic protein obtained from the castor bean, Ricinus communis, Euphorbiaceae. Ricin is a heterodimer comprising an A chain and a B chain joined by a disulfide bond. Ricin A chain (RTA) is an N-glycosidase enzyme that irreversibly damages (i.e. hydrolyzes) a specific adenine base from 28S rRNA. Once the rRNA has been damaged, the cell cannot make protein and will inevitably die (cytotoxicity). As RTA exhibits this type of destructive catalytic activity, RTA is commonly referred to as a type II ribosome inactivating protein (RIP). See Lord, et al. (1991) Semin. Cell Biol. 2(1):15-22. RTA has been coupled with a targeting moiety to selectively destroy target cells such as tumor cells. See U.S. Pat. Nos. 4,80,457; 4,962,188; and 4,689,401; see also Vitetta et al. (1993) Trends Pharmacol. Sci. 14:148-154 and Ghetie & Vitetta (1994) Cancer Drug Delivery 2:191-198.
The toxic consequences of ricin are due to the biological activity of RTA. Ricin B chain (RTB) binds the toxin to cell surface receptors and then RTA is transferred inside the cell where inhibition of ribosome activity occurs. The human lethal dose of ricin toxin is about 1 μg/kg. As highly purified ricin is commercially available, the use of ricin toxin in biological warfare and terrorism is highly possible and probable. Unfortunately, there is no effective antidote for toxic exposure to ricin. Thus, attempts have been made to provide vaccines against ricin intoxication.
Ricin vaccines have been prepared by isolating the natural toxin from castor beans, and treating the toxin with harsh chemicals, typically formaldehyde, to reduce the toxic activity. See Hewetson, et al. (1993) Vaccine 11(7):743-746; Griffiths, et al. (1995) Hum. Exp. Toxicol. 14(2):155-164; Griffiths, et al. (1999) Vaccine 17(20-21):2562-2568; and Yan, et al. (1996) Vaccine 14(11):1031-1038. The first generation vaccines are called “toxoid” vaccines as they are made directly from natural toxin itself. The second generation ricin vaccines comprise wild-type (wt) RTA, but not RTB. The second generation ricin vaccines include deglycosylated RTA (dgRTA) vaccines. The third generation ricin vaccines contain amino acid substitutions in the active site of RTA which result in reduced N-glycosidase activity (e.g Y80A mutations) and/or reduced Vascular Leak Syndrome (VLS) (e.g. Y76M mutations). The fourth generation ricin vaccines contain amino acid deletions and truncations in the RTA which increase the stability of the RTA molecule. See, for example, U.S. Pat. No. 6,869,787.
Since RTA vaccines are limited by their propensity to self-aggregate and denature at or near production and storage temperatures (35-40° C.), a need still exists for ricin vaccines that have a reduced ability to self-aggregate and are thermally stable under production and storage conditions, yet remain therapeutically effective.